Rapid chromogenic detection method for skeletonema costatum

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a rapid chromogenic detection method of skeletonema costatum, a primer group in the rapid chromogenic detection method comprises a primer SC3-F3 with a sequence as shown in SEQ ID NO.1, a primer SC3-B3 with a sequence as shown in SEQ ID NO.2, a primer SC3-FIP with a sequence as shown in SEQ ID NO.3, a primer SC3-BIP with a sequence as shown in SEQ ID NO.4, and primers SC3-LF and SC3-LB with sequences as shown in SEQ ID NO.5-6. Under the condition of ensuring that target sequences are consistent, compared with a conventional PCR (Polymerase Chain Reaction) method, the method disclosed by the invention has the advantages that the amplification specificity and the sensitivity are not lower than those of the conventional PCR technology, the amplification can be completed only at the constant temperature of 60-65 DEG C for 40-50 minutes, the product concentration is high, the amplification effect is stable, and the method is suitable for large-scale popularization and application. Therefore, the method has a good application prospect in the field of rapid detection of red tide algae.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and particularly relates to a rapid color development detection method for Skeletonema costatum. Background Art

[0002] Skeletonema costatum belongs to the phylum Bacillariophyta and the genus Skeletonema. It is a very common floating algae in coastal waters around the world, especially in temperate and subtropical waters. This algae species is distributed in various sea areas in China, often multiplying in large numbers and forming algal blooms. The large-scale proliferation of Skeletonema costatum will not only destroy the balance of the marine ecosystem and affect the survival and reproduction of other marine organisms, but also lead to water quality deterioration and affect the sustainable use of fishery resources. Therefore, strengthening the research on the causes of Skeletonema costatum red tides, dynamic monitoring and early warning mechanism construction has become an important task in marine environmental protection and management.

[0003] At present, the detection of Skeletonema costatum mainly relies on morphological identification under a microscope and high-precision molecular biological analysis technology. Microscopic morphological identification requires the inspector to have a deep knowledge of algae classification and rich practical experience to accurately distinguish algae with similar morphology but different species. However, this process is not only time-consuming, but also easily affected by subjective judgment. Although molecular biology technology has high accuracy and specificity, its high experimental equipment cost, complex sample processing steps and long detection cycle seriously limit the application of this method in field monitoring or emergency response scenarios. Given that the rapid response and effective prevention and control of Skeletonema costatum red tides are of great significance to the protection of marine ecological environment, the maintenance of fishery resources and human health and safety, it is particularly urgent to develop a fast, intuitive, economical and easy-to-operate on-site detection method.

[0004] In summary, in order to overcome the limitations of existing detection technologies and meet the needs of real-time and efficient monitoring of Skeletonema costatum, the present invention aims to provide a rapid colorimetric detection method that does not require complex instruments, is easy to operate, and can instantly display the presence and relative concentration of Skeletonema costatum. Summary of the invention

[0005] The purpose of the present invention is to provide a rapid color development detection method for Skeletonema costatum, which does not require complex instruments and is easy to operate. It can instantly display the presence and relative concentration of Skeletonema costatum, providing strong technical support for early warning and emergency treatment of red tides.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] The present invention provides a primer set for detecting Skeletonema costatum, wherein the primer set comprises a primer SC3-F3 whose sequence is shown in SEQ ID NO.1, a primer SC3-B3 whose sequence is shown in SEQ ID NO.2, a primer SC3-FIP whose sequence is shown in SEQ ID NO.3, a primer SC3-BIP whose sequence is shown in SEQ ID NO.4, a primer SC3-LF whose sequence is shown in SEQ ID NO.5, and a primer SC3-LB whose sequence is shown in SEQ ID NO.6.

[0008] The present invention also provides a kit for detecting Skeletonema costatum, and the kit comprises the primer set.

[0009] Furthermore, the kit also includes Bst DNA polymerase, buffer, dNTPs, magnesium sulfate, double distilled water and hydroxynaphthol blue dye (HNB).

[0010] Furthermore, the kit also includes a lysis solution for extracting algae DNA.

[0011] The present invention also provides a rapid color development detection method for Skeletonema costatum, comprising the following steps:

[0012] 1) After the algae sample to be tested is collected by centrifugation, the supernatant is removed and then resuspended in TE buffer; the suspension is added to a lysis buffer for lysis; the lysis supernatant is added to a diluent for dilution, and the diluent is used as a template for detection;

[0013] 2) adding the template prepared in step 1) to a constant temperature amplification reaction solution containing hydroxynaphthol blue dye and Bst DNA polymerase, using the primer set, reacting at 63° C. for 30-40 minutes, and determining the result by color change;

[0014] 3) The result is determined as follows: if the negative control remains violet and the positive control changes from violet to sky blue with turbidity, the test is normal; if the sample is violet and clear, it is negative; if the sample is sky blue with turbidity, it is positive.

[0015] Furthermore, the diluent is 0.1% Tween20.

[0016] Furthermore, the negative control is prepared by adding double distilled water instead of DNA into the reaction system.

[0017] The present invention also provides an application of the primer set or the kit in detecting Skeletonema costatum.

[0018] Beneficial effects:

[0019] The present invention provides a target sequence designed with the ITS region sequence of Skeletonema costatum as a primer, designs a set of primers that can be used for specific amplification of target algae genes, explains the binding position of the primers and the target sequence, and analyzes the reasons for the failure of primer design. In addition, the selected primers of Skeletonema costatum are specifically verified using purified genomic DNA. At the same time, a conventional PCR method is used to compare the primers with the target sequence under the condition of ensuring the consistency of the target sequence. The results show that the method is not inferior to the conventional PCR technology in terms of amplification specificity and sensitivity, and the method only needs to be kept constant at 60-65°C for 40-50 minutes to complete the amplification, the product concentration is high, and the amplification effect is stable, indicating that it has good application prospects in the field of rapid detection of red tide algae. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is a diagram showing the electrophoresis results of the ITS region amplification of Skeletonema costatum in the present invention;

[0022] Figure 2 This is a comparison result of the ITS region gene sequence of Skeletonema costatum in the present invention;

[0023] Figure 3 This is a diagram showing the alignment results of the ITS-specific segment sequences of Skeletonema costatum in the present invention;

[0024] Figure 4 This is a comparison result of the SSU region gene sequence of Skeletonema costatum in the present invention;

[0025] Figure 5 This is a diagram showing the sequence alignment results of the target algae SSU specific segment of Skeletonema costatum in the present invention;

[0026] Figure 6 This is an illustration of typical amplification results in the present invention;

[0027] Figure 7 This is a primer validation result diagram of Skeletonema costatum in the present invention, wherein PC: experimental group with added genomic DNA; NTC: control group without template;

[0028] Figure 8 Schematic diagram of primer binding sites of Skeletonema costatum in the present invention;

[0029] Fig. 9The figure is the result of primer specificity verification of Skeletonema costatum in the present invention, wherein A: electrophoresis result; B: color development result; M: DL2000 DNA marker; SC: Skeletonema costatum; PM: Prorocentrum microphyllum; PL: Prorocentrum lima; AC: Alexandrium catenella; AP: Alexandrium pacificum; AS: red tide algae; EP: Enteromorpha; PD: Prorocentrum donghaiense; PG: Phaeocystis spherical; KM: Karenia mikimotoi; CC: Chaetoceros spiralis; PT: Phaeochromis triangularis; TP: Thalassiosira pseudomicronaria; NTC: no template control;

[0030] Fig.10 This is a graph showing the primer sensitivity verification result of Skeletonema costatum in the present invention, wherein A: electrophoresis result after detection by the present method; B: electrophoresis result of PCR reaction; C: visualization result after detection by the present method; 1-6: 6 ng / μL, 0.6 ng / μL, 0.06 ng / μL, 0.006 ng / μL, 0.0006 ng / μL, and 0.00006 ng / μL of Skeletonema costatum genomic DNA were added as reaction templates, respectively; NTC: no template control. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Unless otherwise specified, the chemical reagents, biochemical reagents and materials used in the present invention can be obtained from commercial sources.

[0037] Example 1

[0038] 1 Primer design and target sequence analysis

[0039] The ITS region gene of Skeletonema costatum was amplified using universal primers of the ITS region of eukaryotic algae. The electrophoresis results of the amplified products are shown in Figure 1 shown.

[0040] The length of the amplified product of the ITS region of Skeletonema costatum is between 600 and 750 bp, which is similar to the length of the ITS region sequence of the target algae in GenBank. The electrophoresis result shows a single band, and the negative control has no amplification, which can confirm that the amplification is successful. The amplified product is sent to the company for sequencing, and the obtained sequence is shown in SEQ ID NO.7.

[0041] The sequencing results were submitted to GenBank for BLASTn comparison analysis. Figure 2 As shown, the sequencing results have a high degree of identity with the Skeletonema costatum sequence in GenBank, both above 98%, so it can be determined that the sequenced algae species is Skeletonema costatum.

[0042] Then, the ITS region sequences of 13 experimental algae were compared using the software Bioedit, and the specific segment of Skeletonema costatum was selected for the design of primers in this method. The sequence comparison results are shown in Figure 3 shown.

[0043] Similarly, the SSU segment sequence was subjected to conservation analysis, and the results were as follows Figure 4 The SSU region sequence of Skeletonema costatum selected in this example has a small difference between different strains of the same species, in other words, the target sequence has a good conservation.

[0044] The software Bioedit was used to compare the SSU sequences of 13 experimental algae, and the specific segment of Skeletonema costatum was selected for the design of LAMP primers. The sequence comparison results are shown in the figure. Figure 5 shown.

[0045] 2 Primer screening results and final target sequence selection

[0046] The detection results of this method are different from those of conventional PCR reactions. Its multi-primer characteristics and unique amplification mechanism determine the high amplification specificity. The positive amplification product is reflected as a waterfall-like amplification band in the electrophoresis diagram, and the negative product has no amplification band (only primer dimer bands below 100bp). Typical amplification results are as follows: Figure 6 shown.

[0047] For the primers that successfully amplified, the products of the experimental group (adding DNA from Skeletonema costatum) showed waterfall-like bands on the electrophoresis graph, while the products of the negative control group (using an equal amount of double-distilled water instead of DNA) showed no amplified bands on the electrophoresis graph, only primer dimer bands with a length of less than 100 bp (such as Figure 6 Primer groups 1, 5, and 6 in the experiment). There are several types of primer groups that fail to amplify: ① No amplification reaction occurs in the experimental group. The reason why this primer is unusable is that it cannot successfully amplify the target sequence (such as primer group 3). ② Amplification bands appear in both the experimental group and the negative control group. This primer group is relatively common. The reason for this phenomenon may be that the primer sequence is improperly selected, resulting in non-specific amplification of the primers as amplification templates, so amplification also occurs in the negative control group, such as Figure 6 As shown in primer set 4 and primer set 7, it is impossible to determine whether the amplified bands in the experimental group are formed by the primers amplifying the target sequence. In other words, it is impossible to ensure the amplification specificity of the primer set for the target sequence. ③ The amplified bands in the experimental group appear sometimes and disappear sometimes, and there are no amplified bands in the negative control. The main reason for this type of failure is that the primers are not stable enough to amplify the target sequence.

[0048] Based on the above content, the detection system of Skeletonium ribatum was constructed and placed in a PCR instrument for amplification reaction. The product was detected by 1.5% agarose gel electrophoresis. Since this method requires more primers (generally a set of primers consists of 4 to 6 primers), non-specific amplification is easy to occur. After preliminary verification of primer specificity, the designed multiple sets of primers were used for amplification reaction. The reaction system and conditions were as follows: 0.1μM F3 and B3, 0.8μM FIP and BIP, 0.2μM LF, 0.2μM LB, 1.4mM dNTP, 6mM MgSO4, 1×Bst buffer [10mM KC1, 20mM Tris-HCl (pH8.8), 2mM MgSO4, 10mM (NH4)2SO4, 0.1% Triton X-100], 8U Bst DNA polymerase and 2μL template, the total system was 25μL, and the remaining volume was supplemented with double distilled water. The reaction product was detected by 2% agarose gel electrophoresis. The final detection results of the designed primers are as follows: Figure 7 As shown, Figure 7 From left to right in the figure, every three lanes are a set of Skeletonema costatum primers, and the order is: two parallel groups with Skeletonema costatum genomic DNA added, and a negative control group with genomic DNA replaced by double distilled water. The criterion for successful amplification is that the first two lanes (i.e., samples with added DNA templates) produce ladder-like bands, while the third lane (negative control) only has primer dimer bands below 100bp.

[0049] When screening primers, in addition to ensuring that the primers can be successfully amplified (positive control has amplified bands, negative control has no amplified bands), it is also necessary to ensure that the primers have a high amplification efficiency (high product concentration, that is, brighter electrophoresis bands). Based on the above two aspects, the applicant combined Figure 7 According to the amplification results, SC3 was selected as the best primer for Skeletonema costatum, and the primer sequences are shown in Table 1.

[0050] Table 1 Optimal primer sequence information for Skeletonema costatum

[0051]

[0052]

[0053] It can be seen that the primers of Skeletonema costatum are composed of 6 primer sequences, including 4 regular primers and 2 loop primers. In addition to accelerating the amplification reaction rate, the loop primers can also enhance the amplification specificity of the primers. Figure 8As shown. The ITS region gene consists of three parts: ITS1 region gene, 5.8S region gene, and ITS2 region gene. Among them, the 5.8S region sequence is the most conservative, followed by ITS1, and the ITS2 region has the strongest specificity. The primers for Skeletonema costatum finally selected in this embodiment all use the ITS region as the target sequence, ensuring the specificity of the primers.

[0054] 3 Specificity verification results

[0055] In order to ensure the amplification specificity of the Skeletonema costatum primers, the Skeletonema costatum primers were cross-validated using various red tide algae genomic DNA as templates to ensure that the target algae primers can only successfully amplify the algae and will not amplify other red tide algae. By adding an appropriate concentration of hydroxynaphthol blue (HNB) dye to the reaction system, the test results can be visualized. The positive amplification product is sky blue with turbidity, and the negative product is violet and clear. The products were detected using 1.5% agarose gel electrophoresis. The verification results of the Skeletonema costatum primer specificity are shown in the figure below. Fig. 9 shown.

[0056] Depend on Fig. 9 It can be seen that the specific primers for Skeletonema costatum in this method can only achieve amplification with Skeletonema costatum, and waterfall-like step amplification bands appear on the electrophoresis diagram, and the products are mainly concentrated between 100 and 500 bp; the positive amplification product appears sky blue with turbidity, and the negative amplification product appears violet and clear and transparent. The color development results are consistent with the electrophoresis results, which proves that the detection primers for Skeletonema costatum in this method have high interspecies specificity.

[0057] 4 Sensitivity verification results

[0058] The genomic DNA solution of Skeletonema costatum was diluted 10 times in a series of dilutions (6×10 -5 ~6×10 0 ng / μL), and then used it as a reaction template for amplification. The product was detected by 1.5% agarose gel electrophoresis, and the amplification results were visualized by adding HNB dye. At the same time, the PCR method was used for horizontal comparison to verify the results. Fig.10 As shown in Table 2.

[0059] Depend on Fig.10 It can be seen that when the initial template concentration is between 6×10 -4 ~6×10 0 ng / μL, this method can amplify the DNA of Skeletonema costatum and produce waterfall-like amplified bands (such as Fig.10 The corresponding reaction tube is sky blue and turbid (as shown in A). Fig.10 Therefore, the primer sensitivity of Skeletonema costatum in this method can reach 6×10-4 ng / μL genomic DNA.

[0060] Table 2 Results of primer sensitivity test for Skeletonema costatum

[0061]

[0062] Rapid color detection of Skeletonema costatum in Example 2

[0063] 1. Test materials and equipment

[0064] a) Reagents:

[0065] i Amplification reaction system: Bst DNA polymerase, buffer, dNTPs, magnesium sulfate, double distilled water, hydroxynaphthol blue dye (HNB) and primers SEQ ID NO.1-6 designed in Example 1.

[0066] ii Cracking system:

[0067] Lysis buffer composition: 50 mM Tris, 100 mM EDTA, 50 mM NaCl, 1% (w / v) SDS, 80% (v / v) formamide pH 8.0. 1 mL of lysis buffer is pre-loaded in a lysis tube.

[0068] Diluent composition: 0.1% Tween 20. 0.5 mL of diluent is pre-filled in each diluent tube.

[0069] b) Instrument: thermostat

[0070] 2. Reaction system

[0071] The components of the detection system and the concentrations of each component are shown in Table 5, and the total reaction system is 25 μL.

[0072] The specific reaction system in the detection process is shown in Table 3:

[0073] Table 3 Reaction system

[0074]

[0075] Note: The components of the above buffer include: 200mM Tris-HCl, 100mM (NH4)2SO4, 20mM MgSO4, 1% Triton X-100, pH8.8.

[0076] 3. Experimental Procedure

[0077] a) Collection and lysis of algal cells

[0078] The natural seawater containing Skeletonema costatum was filtered with a 0.45 μm pore size filter membrane, and then the filter membrane was rinsed with sterilized seawater to achieve the effect of concentrating and enriching algal cells; the seawater containing Skeletonema costatum was centrifuged at 12000×g for 5 min, and the supernatant was removed and resuspended with 250 μL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), fully shaken, vortexed for 30 s, and 200 μL of the suspension was added to the lysis solution tube for lysis (1 mL of lysis solution was pre-loaded in the lysis tube); 10 μL of the above lysis supernatant was added to the dilution tube and mixed thoroughly (0.5 mL of dilution solution was pre-loaded in the dilution tube). At this time, the liquid in the dilution tube can be used as the detection object.

[0079] b) Detection

[0080] Take several 200 μL centrifuge tubes and prepare the reaction system according to the reaction system formula in Table 3. After the system is prepared, place the reaction system after adding the sample in a thermostat and react at 63°C for 30 to 40 minutes. The result is determined by the color change.

[0081] c) Result determination

[0082] When the negative control is violet and the positive control changes from violet to sky blue, the test process is normal. If the sample to be tested is violet, it is negative; if the sample to be tested is sky blue, it is positive.

[0083] In summary, the present invention uses the ITS region sequence of Skeletonema costatum as the primer to design the target sequence, designs a set of primers that can be used for the specific amplification of the target algae gene, explains the binding position of the primers and the target sequence, and analyzes the reasons for the failure of the primer design. In addition, the purified genomic DNA was used to verify the specificity of the selected set of primers for Skeletonema costatum. At the same time, the conventional PCR method was used to compare it horizontally while ensuring the consistency of the target sequence. The results showed that the method was not inferior to the conventional PCR technology in terms of amplification specificity and sensitivity, and the method only needed to be kept constant at 60-65°C for 40-50 minutes to complete the amplification, the product concentration was high, and the amplification effect was stable, indicating that it has good application prospects in the field of rapid detection of red tide algae.

[0084] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A primer set for detecting Skeletonema costatum, characterized in that: The primer set includes primer SC3-F3 whose sequence is shown in SEQ ID NO.1, primer SC3-B3 whose sequence is shown in SEQ ID NO.2, primer SC3-FIP whose sequence is shown in SEQ ID NO.3, primer SC3-BIP whose sequence is shown in SEQ ID NO.4, primer SC3-LF whose sequence is shown in SEQ ID NO.5, and primer SC3-LB whose sequence is shown in SEQ ID NO.

6.

2. A kit for detecting Skeletonema costatum, characterized in that: The kit comprises the primer set according to claim 1.

3. The kit according to claim 2, characterized in that The kit also includes Bst DNA polymerase, buffer, dNTPs, magnesium sulfate, double distilled water and hydroxynaphthol blue dye (HNB).

4. The kit according to claim 2, characterized in that The kit also includes a lysis solution for extracting algae DNA.

5. A rapid color development detection method for Skeletonema costatum, characterized in that: The following steps are involved: 1) After the algae sample to be tested is collected by centrifugation, the supernatant is removed and the sample is resuspended in TE buffer; The suspension is added to a lysis solution for lysis; the lysis supernatant is added to a diluent for dilution, and the diluent is used as a template for detection; 2) adding the template prepared in step 1) to a constant temperature amplification reaction solution containing hydroxynaphthol blue dye and Bst DNA polymerase, using the primer set of claim 1, reacting at 63° C. for 30-40 minutes, and determining the result by color change; 3) The result is determined as follows: if the negative control remains violet and the positive control changes from violet to sky blue with turbidity, the test is normal; if the sample is violet and clear, it is negative; if the sample is sky blue with turbidity, it is positive.

6. The method according to claim 5, characterized in that The diluent is 0.1% Tween20.

7. The method according to claim 5, characterized in that The negative control is prepared by replacing DNA with double distilled water and adding it into the reaction system.

8. Use of the primer set according to claim 1 or the kit according to any one of claims 2 to 4 in detecting Skeletonema costatum.

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